Negi Vishal Singh, Borthakur Dulal
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East West Road, Honolulu, HI, 96822, USA.
Methods Mol Biol. 2016;1405:59-77. doi: 10.1007/978-1-4939-3393-8_7.
Heterologous expression of eukaryotic genes in bacterial system is an important method in synthetic biology to characterize proteins. It is a widely used method, which can be sometimes quite challenging, as a number of factors that act along the path of expression of a transgene to mRNA, and mRNA to protein, can potentially affect the expression of a transgene in a heterologous system. Here, we describe a method for successful cloning and expression of mimosinase-encoding gene from Leucaena leucocephala (leucaena) in E. coli as the heterologous host. Mimosinase is an important enzyme especially in the context of metabolic engineering of plant secondary metabolite as it catalyzes the degradation of mimosine, which is a toxic secondary metabolite found in all Leucaena and Mimosa species. We also describe the methods used for characterization of the recombinant mimosinase.
在细菌系统中进行真核基因的异源表达是合成生物学中表征蛋白质的一种重要方法。这是一种广泛使用的方法，但有时可能颇具挑战性，因为从转基因表达为mRNA以及从mRNA表达为蛋白质的过程中，许多因素都可能潜在地影响转基因在异源系统中的表达。在此，我们描述了一种成功克隆和表达来自银合欢（Leucaena leucocephala）（银合欢属）的含羞草酶编码基因，并以大肠杆菌作为异源宿主进行表达的方法。含羞草酶是一种重要的酶，特别是在植物次生代谢产物的代谢工程背景下，因为它催化含羞草碱的降解，含羞草碱是在所有银合欢属和含羞草属物种中发现的一种有毒次生代谢产物。我们还描述了用于表征重组含羞草酶的方法。
